Molecular Characterization of Fluoroquinolone-Resistant Aeromonas spp. Isolated from Imported Shrimp

Abstract

bSixty-three nalidixic acid-resistant Aeromonas sp. isolates were obtained from imported shrimp. Phylogenetic analysis of gyrB sequences indicated that 18 were A. enteropelogenes, 26 were A. caviae, and 19 were A. sobria. Double missense mutations in the quinolone resistance-determining region (QRDR) of gyrA at codon 83 (Ser¡Val/Ile) and codon 92 (Leu¡Met) coupled with a point mutation of parC at codon 80 (Ser¡Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of A. enteropelogenes and A. caviae strains harbored toxin genes, whereas only a few A. sobria strains harbored these genes. The fluoroquinolone-resistant Aeromonas spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent Aeromonas spp. to rat epithelial cells. The United States in 2009 imported 589,670 metric tons of farmed shrimp worth more than $6 billion from Asia (27). Copious amounts of antibiotics are used in the shrimp ponds to stimulate growth and to retard the incidence of diseases caused by overcrowded, factory farm conditions (4, 5, 9). The indiscriminate use of these antibiotics may select bacteria resistant to multiple antibiotics, and such bacteria may transfer their antibiotic resistance determinants to pathogenic bacteria (14,16).Aeromonads are ubiquitous, psychrophilic, Gram-negative microbes commonly found in fresh water, estuaries, and other coastal waters (6,7,8). They are known to cause hemorrhagic septicemia in aquatic organisms. These microbes also cause several diseases in penaeid shrimp (5). Antibiotics, such as fluoroquinolones, are used to curtail infections (5, 9). However, prolonged abuse of antibiotics could result in the selection of fluoroquinolone-resistant Aeromonas spp. (20). The presence of fluoroquinolone-resistant aeromonads in shrimp could pose public health concerns because these bacteria are associated with outbreaks of human infectious diseases in immunocompromised patients (9, 29). Quinolones are the drugs of choice for the treatment of Aeromonas-induced infections (11). Thus, U.S. regulatory agencies, such as the FDA and CDC (28), want to limit the prevalence of fluoroquinolone-resistant bacteria in food-producing animals to protect the efficacy of the drugs. Since little information is available on the prevalence of fluoroquinolone-resistant aeromonads in imported shrimp, we decided to investigate the prevalence of fluoroquinolone-resistant Aeromonas spp. and the mechanism of resistance in these bacteria to the antibiotic. We report here for the first time that imported shrimp may be a reservoir of virulent, fluoroquinolone-resistant strains of A. enteropelogenes, A. caviae, and A. sobria.Isolation, characterization, and identification of bacteria. Bacteria were isolated from frozen shrimp (Penaeus monodon) imported from Thailand. Typically, 2 g of thawed shrimp sample was enriched for 6 h in alkaline peptone medium (pH 8.0) supplemented with 30 g/ml of ampicillin and nalidixic acid. Enriched samples were streaked on MacConkey agar and incubated at 37°C overnig...

Development of a PCR Assay Based on a Single–Base Pair Substitution for the Detection of Aeromonas caviae by Targeting the gyrB Gene

“…In the case of ACF2-ACR, approximately 90% positive results were obtained with A. caviae, a rate that may be due to nucleotide changes in a few isolates. Some reports have indicated that mutations in the gyrB gene are not frequent and that when they occur they are often attributable to quinolone resistance (Goni-Urriza et al 2002;Shakir et al 2012).…”

Unique Features of Aeromonas Plasmid pAC3 and Expression of the Plasmid-Mediated Quinolone Resistance Genes

“…The presence of fluoroquinolone resistance genes, including qnrA , qnrB , qnrC , qnrD , qnrS , qnrVC1 , qnrVC4 , qepA , aac ( 6′ ) -Ib-cr , oqxA , and oqxB ( 49 , 52 – 54 ), and mutations in the QRDRs of the gyrA , gyrB , and parC genes ( 17 , 18 ) were verified by PCR amplification and sequencing as previously described (see Table S4 ).…”

“…The presence of quinolone resistance determinants has been reported in various Aeromonas spp. ; several strains contain multiple resistance mechanisms, including mutations at quinolone resistance-determining regions (QRDRs) ( 17 – 21 ), efflux pumps ( 18 , 21 , 22 ), and PMQR genes ( 23 – 26 ). In some Aeromonas sp.…”

Characterisation of novel mutations involved in quinolone resistance in Escherichia coli isolated from imported shrimp

“…Detection and amplification of the quinolone resistance genes were carried out as detailed elsewhere [19,20].…”